Brain metastases occur in approximately 15% of metastatic breast cancer patients and confer a dismal prognosis. Brain metastases are thought to increasing, particuarly among metastatic patients with Her-2+ or triple negative tumors. Our goals are to identify genes that functionally contribute to brain metastatic progression and to identify and validate preclinical leads. A murine preclinical model of brain metastasis was developed using a derivative of the MDA-MB-231 breast carcinoma cell line (231-BR cells). The proliferative and apoptotic rates exhibited by this cell line, as well as the presence of a neuro-inflammatory response, closely correlate with data from 16 resected brain metastases of human breast cancer, suggesting that the model is relevant. Additional models of brain metastasis of breast cancer have been estalblished to provide a measure of heterogeneity including the murine 4T1 mammary cell line (4T1-BR5) and Her-2 transfected human MCF-7 (ER+) cells (MCF-7-Her-2-BR3). In collaboration with Drs. Quentin Smith and Paul Lockman, Texas Tech University, the permeability of experimental 231-BR and 4T1-BR5 metastases was quantified. In press collaborative research showed that experimental brain metastases of breast cancer were heterogeneous in their permeability, both within and between metastases in the same brain. While most metastases were permeable as compared to the normal brain blood-brain barrier, 10% exhibited sufficient permeability to enable a cytotoxic response to a systemic drug. The data argue that a new class of inherently brain-permeable drugs will be needed for this complication. We tested the hypothesis that Her-2 overexpression alters the natural history of breast cells to render them more brain metastatic. Her-2 transfectants of the 231-BR cells produced three fold greater large brain metastases, proportional to MRI detectable metastases in a human brain. We determined the efficacy of the dual EGFR/Her-2 tyrosine kinase inhibitor, lapatinib, on the brain metastatic colonization of a human breast carcinoma cell line.). Lapatinib prevented the brain colonization of 231-BR-Her-2 cells by 53% and inhibited the phospho-Her-2 staining of treated brain metastases in vivo, demonstrating that the drug hit its target. Rational combinations with lapatinib are under investigation to improve its preventive efficacy. We investigated the effect of pazopanib, a VEGFR, PDGFR and c-kit inhibitor. Pazopanib was anti-proliferative to 231-BR-Her-2 cells as well as endothelial cells. Inhibition of pErk was observed in the tumor cells despite the fact that they harbored mutations in both Ras and B-Raf. Enzymatic assays demonstrated that pazopanib has B-Raf inhibitory activity distinct from previously reported B-Raf inhibitors. It is preferentially inhibitory to Her-2 activated wild type B-Raf, as compared to the common V600E melanoma mutation. In vivo pazopanaib significantly prevented the brain metastatic colonization of both 231-BR-Her-2 and MCF-7-Her-2-BR3 cells. Analysis of the treated brain metastases showed a dimunition of pErk and PMek tumor cell staining, with no dimunition of blood vessel density, suggesting that the B-Raf inhibitory activity of pazopanib was functionally important. Lapatinib/pazopanib combination experiments are underway. In collaboration with Dr. George Sledge, Indiana University, a 13-gene signature was developed from gene expression profiling of primary tumor material from metastatic Her-2+ breast cancer patients, that predicts short ( less than 2 years) versus long ( greater than 3 years) time to the development of brain metastases. Included among the 13 genes were the overexpression of three DNA double strand break (DSB) genes, Rad51, Bard1 and FancG, all overexpressed in tumors rapidly developing brain metastases. We have asked whether Rad51 and Bard1 overexpression has a phenotypic effect on normal MCF-10A human breast cells, analogous to overexpression in a primary tumor. Using 3D cultures, an increase in invasion was observed with Rad51 overexpresssion. Functional primary tumor and brain metastasis studies are underway to further define this pathway. Confirmatory studies on the 13 gene signature are underway at IU. A microarray analysis of surgically resected brain metastases of breast cancer, using laser capture microdissection, amplification and 30K cDNA arrays. These data were compared to a cohort of unmatched primary breast tumors, matched for histopathology, TNM and grade. A heat map comparing gene expression differences between brain metastses and unmatched primary tumors has been compiled and expression trends validated by QRT-PCR using an independent cohort. Of the genes validated, experiments are ongoing for hexokinase 2 (HK2 and pigment epithelium derived factor (PEDF). PEDF is a secreted protein with anti-angiogenic, tumor suppressor and neuronal viability properties, possibly mediated through multiple receptors. Overexpression of PEDF in the 231-BR cell line reduced the number of large parenchymal metastases significantly. Current experiments are using an intracranial implantation system to determine the effect of PEDF on neuronal viability. Analysis of the gene expression data indicated that 80% of the differentially expressed genes were down-regulated in the brain metastases. We asked whether a HDAC inhibitor could restore gene expression using the 231-BR cell line.Vorinostat was selected from the multiple HDAC inhibitors since its structure appeared favorable for brain penetration. Treatment of mice with 100 mg/kg vorinostat qd, beginning on day five postinjection, significantly reduced the number of large brain metastases. In contrast to the literature, we find no effect of vorinostat on brain histone acetylation as determined by IHC, nor on apoptotic rates. We did uncover a novel mechanism of action, the induction of DNA double stand breaks. These data have been extended by Dr. Kevin Camphausen who observed a super-additive interaction of radiation and vorinostat on intracranial 231-BR implants. A clinical trial of Vorinostat and radiation has opened at Thomas Jefferson University.